Bacteria of the Rhizobiaceae family are gram negative and able to form nitrogen-fixing relationships with legumes. The surface polysaccharides, including the lipopolysaccharides (LPSs), have been shown to play important roles in the symbiotic infection process.
Rhizobium and Bradyrhizobium LPS, as with others, have three structural regions: the lipid A, core oligosaccharide, and O-chain (or O side chain) polysaccharide. The rhizobial O-chain polysaccharides are highly variable and contain many methylated and deoxy glycosyl residues. See Stryer, Biochemistry, 2d Ed., W. H. Freeman and Co., New York, p. 74 (1981).
Before the discovery of the lipid A component of the LPS the term "endotoxin" was used to generically describe the effect of the LPS. The endotoxin from gram(-) bacteria is heat-stable, cell-associated, pyrogenic and potentially lethal. Lipid A is the causative agent of disorders such as septic or toxic shock and is related to other disorders such as Lyme disease.
The lipid A from enteric bacteria is somewhat variable. However, it is generally recognized that such a lipid A consists of a .beta.-1,6-linked glucosamine disaccharide substituted at positions 4' and 1 by phosphomonoester groups. Fatty acids are linked to the hydroxyl and amino groups of the disaccharide to confer hydrophobicity to the lipid A. Also present in enterobacteria are amide and ester-linked D-3-hydroxy fatty acids, which consist of 14 carbons, e.g. O-hydroxymyristic acid. The C3--OH positions of these fatty acids may be further esterified with saturated fatty acids.
Despite these general characteristics, a degree of microheterogeneity occurs among diverse genera and species. Thus, Neisseria species produce 12 carbon 3-hydroxy fatty acids, saturated fatty acid substitution varies and the C'4-phosphoglucosamine disaccharide may contain a 4-amino-L-arabinose in salmonellae and P. aeruginosa as opposed to E. coli and Shigella. A very potent and toxic lipid A is a hexaacyl-1-4'-diphospholipid A. Structurally, a lipid A with one fewer or one more fatty acids will result in a biologically active, yet less toxic moiety. Removal of all fatty acids, however, deprives a particular lipid A of any biological activity. In addition, removal of either phosphate group results in significant loss of toxicity without loss of adjuvant activity. See Zinnser, Microbiology, 20th Ed., Appleton & Lange, Norwalk, Conn., pp. 84-86 (1992).
As discussed above, the cell associated, heat stable toxin of gram-negative bacteria is the lipopolysaccharide (LPS). While both the O-antigen and the core regions modulate the toxic activity of the LPS, it is the lipid A region that possesses the biological activity of the endotoxin (26,31). The structure of the lipid A from enteric bacteria (e.g. E. coli) is shown in FIG. 1. This structure is found in many gram-negative bacteria, and is the minimum structure required for toxic activity. Structural variations of this molecule that lack any one of the substituent groups; e.g. lacking a phosphate or fatty acyl substituent; are less toxic or not toxic (26,31). In addition, the minimal structure for viability of the bacterium requires the addition of two Kdo residues to C-6 of the terminal glucosamine residue (26).
In recent years, workers have discovered that endotoxin induced shock is caused by the ability of the LPS to stimulate host cells, such as macrophages, to produce excessive levels of cytokines (3,12,23). It is the excessive production of these cytokines, e.g. tumor necrosis factor (TNF) and interleukin I (IL-1), that results in toxic shock. At the present time it is probable that macrophages respond to lipid A by two possible mechanisms. The first mechanism involves the interaction of lipid A with a receptor on the macrophage cell surface which results in the release of signals that stimulate the synthesis of cytokines. This mechanism occurs with relatively high concentrations (nM) of lipid A (20,25). The second mechanism involves the binding of the lipid A (or LPS) by a serum protein called the LPS binding protein (LBP). This LPS-LBP complex then interacts with a receptor (CD14) on the surface of the macrophage resulting, again, in the production of signals with stimulate the synthesis of cytokines (20,30,34,35,42). This second mechanism is active at low lipid A concentrations (pM) (20).
The potent biological activity of lipid A has directed numerous research efforts toward developing useful applications of this activity. First, the necessity of a minimal structure for bacterial viability has led workers to synthesize compounds which inhibit the synthesis of this structure, and thereby, act as a new class of antibiotics (15,16). These inhibitors are based on their ability to inhibit Kdo synthase activity. Second, the ability of lipid A to stimulate the immune system has resulted in the investigation of the use of lipid A, and modified lipid A structures and analogs, as therapeutic anti-tumor agents (33,36), and, more recently, as adjuvants for vaccine development (1). Third, therapeutic agents which inhibit the interaction of lipid A with macrophages have been investigated as treatments for sepsis (13). These agents, have been polyclonal or monoclonal antibodies against common structural regions of lipid A (the core oligosaccharide or lipid A) (6,7,11,13,18,28,38,41,44,45), monoclonal antibodies against the LBP or CD14 proteins (2,11), and lipid A analogs which inhibit the binding of lipid A to LBP or CD14 (17,32). The use of antibodies in animal studies has warranted their testing in humans. Three different trials have given inconsistent results. However, in a subset of patients with gram-negative sepsis the results seemed to be beneficial and safe (13). The overall draw-back of this type of therapy is the high cost of acquiring these antibodies combined with the marginal benefits (as obtained in the recent clinical trials). Another useful approach is the use of lipid A analogs as antagonists for the toxic activity of lipid A. Several synthetic compounds have been examined (14,21,26,27,31,37), however the compound with the most potential is based on the lipid A from Rhodobacter sphaeroides (17,32) and on that from Rhodobacter capsulatus (FIG. 2) (19). This lipid A, which is unusual in that in contains unsaturated and 3-oxo fatty acyl residues, is not toxic and is a potent inhibitor of the ability of lipid A to stimulate cytokine production in an in vitro assay (17,22,32). Recently, a synthetic analog of this compound has been developed by Eisai (10) (FIG. 3) which is an even more potent lipid A antagonist.
The biological responses to LPS/Lipid A challenge are varied. Endotoxin is a potent pleiotropic biomodifier. Response to endotoxin challenge is species, dose, site, and route dependent. Even small doses of lipid A cause extreme changes in body temperature, hematology, immunology, and endocrinology of the subject. Lethal doses lead to hypotension, disseminated intravascular coagulation, irreversible shock, and, ultimately, death.
Most animals exhibit neutropenia and rapid induction of fever and hypotension upon challenge with lipid A from gram(-) bacteria. Intracerebral dosage of endotoxin requires a significantly reduced quantity for similarly devastating results. The most sensitive animals to endotoxin are humans. For instance, only about 2 ng LPS/kg from Salmonella abortus equi induces granulocytosis, a 7-hour fever of about 2.degree. C. maximal temperature rise, and increased plasma cortisol levels. As opposed to the biphasic fever curve in other animals, the human fever response is monophasic. A dose of about 100 .mu.g LPS is lethal in humans.
Known hematologic responses to LPS injection include production of cytokines such as Interleukin-1 (IL-1), Interleukin-6 (IL-6) and tumor necrosis factor (TNF). Significant release of endotoxin into the circulatory system leads to disseminated intravascular coagulation. The Schwartzman reactions are classic examples of endotoxin induced clotting responses. See Zinnser, supra at p. 86.
Lipid A is cleared from the host when human peripheral blood monocytes and neutrophils begin to deacylate the lipid A with an acyloxyacyl hydrolase which removes fatty acids esterified to .beta.-hydroxymyristate acid esters. This deacylation results in significant reduction in toxicity of the resulting modified lipid A. The deacylated lipid A does, however, retain some adjuvant activity and ability to modulate or antagonize further response to LPS.
Current treatment for lipid A challenge includes the use of polymyxin B. Polymyxin B is thought to form a complex with LPS and thereby prevent the toxin from acting. In addition, monoclonal antibodies to tumor necrosis factor may be helpful. Although such treatments are helpful in alleviating some of the devastating effects of lipid A toxicosis, they do not constitute a completely safe and effective treatment. Therefore, there still exists a need for novel, effective treatments for lipid A associated disorders. In addition, there exists a need for a lipid A which is a potent adjuvant without the related toxicity. Finally, there exists a need for a lipid A which can be used to treat or prevent LPS associated disorders. The present invention provides the discovery that the lipid A from Rhizobium leguminosarum biovar phaseoli CE3 satisfies these needs.
There are two reports which describe a lipid A structure from two different strains of R. leguminosarum bv. trifolii (52,53). These reports provide incorrect structures for lipid A. Both of these reports describe structures which differ from each other, and which differ significantly from the structures described herein. Furthermore, the beneficial activities of the lipid A of this invention were not described, e.g., the use of this novel lipid A and its analogs as therapeutic agents to stimulate the immune system, as adjuvants for vaccines, and as lipid A or LPS antagonists to prevent or treat sepsis.
Polymixin B is a cationic cyclic peptide which acts by binding the anionic endotoxin. This antibiotic has been used to remove endotoxins from biochemical preparations so the preparations could be used for in vivo studies. Polymixin B-agarose affinity material is produced for that purpose (Detoxi-Gel.TM., Pierce Chemical Company). Biochemical preparations can be passed through Polymixin B-agarose affinity material and any endotoxin in the preparation will bind to the Polymixin B-agarose affinity material. The Polymixin B-agarose affinity material can be regenerated by removing bound endotoxin with 1% deoxycholate (DOC) solution. The Polymixin B-agarose affinity material can then be reused.